Indian Journal of Experimental Biology
Vol.50, August 2012 pp. 517-530
Radiomodulation by Hoechst 33258 against radiationinduced damage in murine splenocytes
Zubaida Khatoon* & R K Kale†
Free Radical Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India
Received 21 November 2011; revised 7 May 2012
In this study modulatory effect of Hoechst 33258 on radiation induced membrane related signaling events which
ultimately leads to apoptosis has been investigated. Splenocytes from swiss albino mice were irradiated in air at room
temperature in a gamma chamber (240 TBq 60Co Model 4000 A) at the dose-rate of 0.052 Gys-1. Membrane lipid
peroxidation, fluidity, specific activities of antioxidant enzymes, levels of nitric oxide, glutathione and apoptosis in presence
and absence of different concentrations of Hoechst 33258 has been assayed. DNA binding activity of nuclear factor kappa
B and activator protein–1 was also assayed by electrophoretic mobility shift assay. Modulatory effect of Hoechst 33258 was
examined at 3 and 5 Gy using different concentrations (10, 20 and 30 µM). Hoechst 33258 was found to inhibit radiation
induced peroxidative damage and fluidity and lowered the level of nitric oxide and apoptosis - as evident by DNA ladder
assay and FACS, indicating free radicals scavenging potential. Dot plot diagramme clearly showed that 30 µM Hoechst
33258 caused 14% and 19% decrease in apoptotic cells at 3 Gy and 5 Gy of radiation respectively (compared to irradiated
control group). Further DNA binding activity of nuclear factor kappa B and activator protein–1 was also inhibited but the
antioxidant potential of the cells was enhanced. These findings support that Hoechst 33258 protects the cell from undergoing
apoptosis. Hoechst 33258 may have interacted and has an ability to protect splenocytes against radiation induced apoptosis
through modulation of membrane-related signaling events and antioxidant status.
Keywords: Antioxidant enzymes, FACS, Hoechst 33258, Membrane fluidity, Peroxidative damage, Radioprotector
Cellular damage by ionizing radiation is
predominantly mediated through free radicals and
resultant reactive oxygen species1,2. Interaction of
ionizing radiation with water as major cellular
constituent results in generation of primary water
radical species e.g. e-aq, HO., O2.-, H2, H2O+. Direct
action of majority of primary radicals to
bio-molecules are limited due to their short life time
and hence its inability to diffuse up to target
molecule. These primary radicals generated during
water radiolysis react with molecules like oxygen
producing secondary radicals (HO2· , RO2· ), which are
not only relatively stable but may also diffuse to vital
cellular targets like DNA, proteins and membrane3.
Biological membranes play an important role
in radiation-induced cell injury and death4-6.
The membrane organization is an initial step in
————————
*
Correspondent author
Telephone: 91-191-2582003
E-mail: [email protected]
†
Present address: Vice-Chancellor Central University of Gujarat,
Camp Office, 95/1, Sector-2A, Gandhinagar 382 007
Ahmadabad, India
Telephone: 91-79-29289056; Telefax: 91-79-23225742
triggering apoptosis7. A correlation was found
between unrepaired membrane damage and loss of
colony forming ability in cells8-10, the breakdown of
nuclear membrane and chromosomal condensation
and damage to cell11, organization of mitotic spindles,
interphase death in non-proliferating cells and
disorganization of membrane12. Thus the integrity of
membrane is essential for normal functions of the cell,
and the damage, if not repaired, may lead to cell death.
It may be mentioned that radiation-induced alterations
in the structure and function of cellular membrane are
suggested to serve as a signal to activate component
of signal transduction pathways involved in
apoptosis4,6. Further, radiolytically generated free
radicals in plasma membrane and subsequent
oxidative stress also suggested to be an important
component of signaling process leading to
apoptosis. Once triggered, apoptosis proceeds via a
cascade of events that is accomplished in few
hours independently of triggering agent, e.g.,
radiation vs drugs13.
Hoechst 33258 and its ethoxy derivative Hoechst
33342 were shown to have radioprotective ability14-20,
as well as radiosensitizing property21-23. Differential
518
INDIAN J EXP BIOL, AUGUST 2012
radiomodifying property of Hoechst 33258 make it a
possible tool for radiation therapy of cancer.
Radiosensitization is suggested to be mediated by
inhibition of topoisomerase 1 and II22,24,25 and
formation of head-to-head adduct (head-to-head bisbenzimidazoles) which is toxic to cells26. Since tumor
cells and tissues are often associated with reduced
oxygen tension and elevated levels of topoisomerases,
Hoechst 33258 has a higher probability of inducing
radiosensitization in tumor cells and tissues.
Characteristically poor penetration of Hoechst 33258
through cell layers has potential advantage for
preferential protection of normal tissues27, an efforts
are being made to understand the mechanism of
protection by this class of compounds. Hoechst 33258
and its derivatives are known to interact and provide
the stability to DNA leading to radioprotection both in
purified DNA systems, and in cell culture
studies15,16,19,20. It is quite possible that Hoechst 33258
and its derivatives may also interfere with radiationinduced events involved membrane damage and in
turn provide the protection. However, no information
is available on the chemical-pathways related to
membrane events responsible for its protection.
In the present work, modulation of membrane
damage by Hoechst 33258 using splenocytes of
Swiss albino mice has been investigated. An attempt
has also been made to examine the influence of
Hoechst 33258 on apoptosis, DNA binding activity of
an oxidative stress responsive transcription factor
NF-kB
(nuclear
factor
kappa
B),
AP1
(activator protein–1), nitric oxide (NO.) as well as
antioxidant status in irradiated splenocytes.
Findings of this study suggested that Hoechst 33258
has an ability to protect biological system against
radiation effects through modulations of membrane
related events and inhibition of apoptosis.
Materials and Methods
Chemicals—Reduced
nicotinamide
adenine
dinucleotides (NADH), pyrogallol, 1-chloro-2,4dinitrobenzene (CDNB), RPMI-1640 medium
(Roswell Park Memorial Institute medium), FBS
(Fetal Bovine Serum) 2,6-dichlorophenolindophenol
(DCPIP), reduced glutathione (GSH), 1,6 diphenyl1,3,5-hexatriene (DPH), thiobarbituric acid (TBA),
bovine serum albumin (BSA), 5,5-dithio-bis
(2-nitrobenzoic acid (DTNB), N-(1-naphthyl)thylenediamine (NEDD), sulfanilamide, agarose
sodium dodecyl sulphate (SDS), boric acid,
acrylamide, dithiothreitol (DTT), phenylmethanesulfonyl
fluoride (PMSF), leupeptin and aproteinin, apoptosis
detection kit, Hoechst 33258 (4-(6-(4 methylpiperazin-1-yl)1H,3’H-2,5’-bibenzo[d]imidazol-2’-yl)phenol (Fig. 1)
were from Sigma Chemical Co. (St Louis, MO, USA).
NF-kB, AP1 oligonucleotides were obtained from
Bangalore Genei Pvt Ltd, (Bangalore, India).
All other chemicals used were of analytical grade.
Animals—Swiss albino female mice (6-8 weeks old)
maintained in the animal house of the university were
used. Standard feed (Hindustan Lever Ltd, Mumbai,
India) and water were provided ad libitum.
Animal experiments were conducted according to
the ethical guidelines of the Committee for Control
and Supervision of Experiments on Animals,
Government of India.
Irradiation of splenocytes with γ—rays-Splenocytes
from Swiss albino mice suspended in 1×PBS for
enzyme assay and in RPMI-1640 medium for rest of
the experiments, were irradiated in air at room
temperature in a gamma chamber (240 TBq 60Co
Model 4000 A) obtained from the Isotope Division,
Bhabha
Atomic
Research
Centre
(BARC)
(Mumbai, India). The dose-rate used was 0.052 Gys-1
and determined by Fricke’s dosimetry28,29.
Preparation of samples—Briefly, animals were
killed by cervical dislocation and the spleens removed
and washed in PBS. Single cell suspensions were
made in RPMI-1640 by crushing the spleen in
between frosted slides. 5 µl of it were kept aside for
protein determination. Viability of these cells with
used concentrations (10, 20, 30 µM) of Hoechst
33258 was determined by trypan blue exclusion
method both in the medium and in PBS. Cells were
incubated in RPMI-1640 with 1% FBS (v/v) in
presence and absence of different concentrations
(10, 20, 30 µM) of Hoechst 33258 on ice for 30 min
before irradiation20. They were used as such for
assaying lipid peroxidation (LP), fluidity and
AP-1 immediately after irradiation; DNA binding
activity of NF-kB and FACS after 30 min
post-irradiation. For DNA ladder assay and
Fig. 1—Chemical structure of Hoechst 33258
KHATOON & KALE: RADIOMODULATION BY HOECHST 33258
NO. determination 5% FBS (v/v) was added in
splenocytes (treated with different concentration of
Hoechst 33258 in RPMI-1640 without FBS)
immediately after irradiation and then post-irradiation
incubation was done for 6 h and 24 h respectively. All
postirradiation incubation was done at 37 οC in 5% CO2.
For the assay of antioxidant enzymes, the cells
were made and incubated in PBS in presence of
different concentrations (10, 20, 30 µM) of
Hoechst 3325820 on ice for 30 min before irradiation.
Cells were sonicated at a peak-to-peak amplitude of
18 µ immediately after irradiation and centrifuged
thereafter at 16,060 g to remove the debris.
Supernatant was collected and used for measurement
of specific activities of GST, SOD, catalase and DTD.
For GSH content cells were incubated with different
concentrations (10, 20 and 30 µM) of Hoechst 33258
in RPMI-1640 without any serum, irradiated and
assay was done immediately after irradiation.
Determination of specific activity of superoxide
dismutase (SOD)—Superoxide dismutase (SOD) was
assayed by the method of Marklund and Marklund30
by measuring inhibition of pyrogallol at 420 nm.
Assay mixtures (1 mL) contained 0.05 M sodium
phosphate buffer (pH 8.0), 0.1 mM EDTA and
0.27 mM pyrogallol. Reaction was initiated by
addition of enzyme sample, which was pretreated
with Triton X-100 on ice for 30 min. One unit of
enzyme was defined as the amount of SOD required
to produce half-maximal inhibition of auto-oxidation
of pyrogallol.
Determination
of
specific
activity
of
DT-diaphorase (DT-D)—DT-diaphorase (DT-D) was
assayed according to Ernster et al.31 by measuring
reduction of 2,6- dichlorophenolindophenol (DCPIP)
at 600 nm with some modifications. The reaction
mixture contained 50 mM Tris-HCl buffer (pH 7.5),
0.5 mM NADH, 10 µM DCPIP, 0.08% Triton
X-100 and the enzyme sample in a final volume of
1 mL. The reaction was started at 25 οC by addition of
NADH and the activity was calculated using an
extinction coefficient 21 mM /cm. One unit of enzyme
activity was defined as the amount of enzyme
required to reduce 1 µ mole DCPIP/min.
Determination of specific activity glutathioneS-transeferase
(GST)—Glutathione-S-transeferase
(GST) was assayed using the method of Habig et al.32,
by measuring the formation of GSH-CDNB
(1-chloro-2, 4-dinitrobenzene) conjugate at 340 nm.
Reaction volume (1 mL) contained final
519
concentrations of 0.1 M sodium phosphate buffer
(pH 6.5), 1 mM CDNB in ethanol and 1 mM GSH.
The reaction was initiated by the addition of the
enzyme sample. The specific activity was calculated
using an extinction coefficient 9.6 mM/cm and
expressed in terms of µ mole CDNB-GSH conjugate
formed min/mg protein.
Determination of specific activity of catalase—
Catalase was assayed according to method of Aebi33,
by measuring the decomposition of H2O2
(Hydrogen peroxide) to give oxygen and water.
The supernatant was treated with Triton X-100 (25 %)
for 30 min on ice. Then ethanol (10 µl/ml) was added
and again kept for 30 min on ice. The treated
supernatant was added to the assay mixture which
contained 0.1 M sodium phosphate buffer (pH 7.0), 10
mM H2O2 and decrease in absorbance was measured
at 240 nm. The activity was calculated using
extinction coefficient 0.04 mmole/cm. One unit of
catalase activity was defined as amount of enzyme
required to decompose one mole of H2O2 /min.
Determination of membrane fluidity—A 2 mM
solution of 1, 6-diphenyl-1, 3, 5-hexatriene (DPH)
was prepared in tetrahydrofuran and 100 µL of it was
added to 100 mL of rapidly stirring Tris-HCl buffer
(10 mM, pH7.4). Treated and irradiated cells (in
RPMI-1640 with 1% FBS) were counted and
incubated with 2 mM DPH for 30 min at room
temperature
immediately
after
irradiation.
Fluorescence polarization was measured by excitation
with vertically polarized monochromatic light at
365 nm and emission intensity detected at 432 nm
through a polarizer oriented either parallel or
perpendicular to the direction of the polarized excitation
light. The degree of fluorescence polarization was
calculated according to Haggerty et al.34:
Ivv - Ivh (Ihv / Ihh)
P=
Ivv + Ivh (Ihv / Ihh)
where I is the corrected fluorescence, and v and h
indicate values obtained with vertical or horizontal
orientation, respectively, of the excitation and
analyser polarizer in that order. Fluorescence was
monitored on a Shimadzu RF-540 fluorescence
spectrophotometer. Because fluidity was inversely
related to polarization of the probe, membrane fluidity
was expressed as the reciprocal of polarization (1 / P).
Estimation of membrane lipid peroxidation (LP)—
Membrane lipid peroxidation was estimated
520
INDIAN J EXP BIOL, AUGUST 2012
spectrophotometrically by the thiobarbituric acid
(TBARS) method as described by Varshney and
Kale35 and was expressed in terms of TBARS formed
mg/protein. To avoid interference due to spontaneous
peroxidation of membrane and because sufficient
amount of TBARS was formed, the assay was
performed immediately after irradiation. In brief,
0.5 mL cell suspension (treated and irradiated as
explained in section 2.4) was mixed with 1.6 mL
Tris KCl (0.15 M KCl + 10 mM Tris-HCl, pH 7.4)
buffer to which 0.5 mL 30% TCA was added. Then
0.5 ml, 52 mM, TBA (thiobarbituric acid) was added.
The tubes were covered with aluminium foil and
placed in a water bath for 45 min at 80 οC, cooled and
centrifuged at room temperature for 10 min at
14000 g in a REMI-T8 table-top centrifuge.
Absorbance of the clear supernatant was measured
against reference blank of distilled water at 531.8 nm
in spectrophotometer (UV-160).
Protein determination—Protein concentrations
were determined by method of Bradford36, using
bovine serum albumin (BSA) as standard.
Determination of non-protein sulphydryl content—
Non-protein sulphydryl content was determined by
the method described by Moron et al.37 in splenocytes
immediately post-irradiation, using 0.6 mM DTNB
[5, 5-dithio-bis (2-nitrobenzoic acid]. The absorbance
was read at 412 nm and the sulphydryl content
calculated with the help of a standard graph made by
using different concentrations of reduced glutathione
and expressed in terms of µmol/g protein. It was
shown that the sulphydryl content determined by this
assay using DTNB consisted mainly of reduced
glutathione (GSH)38.
Determination of nitric oxide levels—A total of 105
cells were treated as explained in section 2.4 and
irradiated in RPMI-1640 (cells were counted in
RPMI-1640 and 5% FBS (v/v) was added
immediately after irradiation). They were then
incubated at 37 οC in 5% CO2 for 24 h. This
post-irradiation time was chosen as the time point for
the assay after careful time course studies because a
good amount of NO. was detectable at this time
interval (data not shown). After that, cells were
pelleted and nitric oxide levels determined in the
supernatant by the method of Griess39 with some
modifications. Briefly, to 100 µl of the supernatant, an
equal volume of Griess reagent (1% sulfanilamide,
0.1% naphthalene-ethylene diamine dihydrochloride
(NEDD) in 5% orthophosphoric acid) was added and
immediately mixed. After 5 min, 200 µl of the above-
formed product was transferred to a 96-well
flat-bottomed plate and read at 550 nm in a microplate
reader. The nitrite content of each sample was
evaluated from the standard curve made with sodium
nitrite (obtained after linear regression), and was
expressed in µM.
Nucleosomal ladder formation assay for
apoptosis—Internucleosomal DNA fragmentation was
determined by electrophoresis according to the
method of Barry and Eastman40. Cells were incubated
with or without different concentrations of Hoechst
33258 in RPMI for 30 min on ice and irradiated with
gamma-radiation (5% FBS (v/v) was added
immediately after irradiation). In time-course studies,
a clear ladder was observed at 6 h after irradiation
compared with earlier time points (data not shown).
Therefore, the treated and irradiated cells were then
incubated at 37 οC under 5% CO2 for 6 h in RPMI.
A total of 2% agarose in Tris-borate EDTA buffer
was poured into a horizontal gel support. Once the gel
solidified, the section of the gel immediately above
the comb was removed by cutting along the top side
of the comb with a scalpel and filling with
1% agarose, 2% SDS and 64 µg/mL proteinase K. A
total of 106 cells were centrifuged to remove the
medium and resuspended in loading dye and RNAse
A (1:1 by volume). It was loaded directly into the
wells and electrophoresis carried out at 21 V for 16 h
at room temperature. The gel was visualized
under illumination after staining with 2 µg/ml
ethidium bromide.
FACS analysis—Apoptotic cells were detected
based on the principle of Annexin V binding to
translocated plasma membrane PS (Phosphatidyl
serine). During the apoptotic process, PS translocates
from the inner membrane to the outer membrane of
the cells41. FITC-labeled (Fluorescein isothiocyanate)
Annexin-V was added to cultured cells and bound to
exposed PS. FITC signals were detected by flow
cytometry. PI (Propidium iodide) was added to
cultured cells to identify the loss of integrity of the
cell membrane, which is specific for necrotic cells.
Briefly, treated and irradiated cells in RPMI-1640
with 1% FBS (v/v) (as explained in section 2.4) were
incubated for another 30 min in CO2 incubator at
37 οC, subsequently washed with PBS, resuspended in
1 × binding buffer (100 mM HEPES / NaOH, pH 7.5
containing 1.4 M NaCl and 25 mM CaCl2) at a
concentration of approx. 1 × 106 cells/mL. In 500 µL
of the cell suspension 5 µL of the Annexin V-FITC
and 10 µL of propidium iodide was added, incubated
KHATOON & KALE: RADIOMODULATION BY HOECHST 33258
for 10 min and protected from light. After incubation
fluorescence of the cells was determined immediately
with a flow cytometer (BDLSR, Becton Dickinson
California). The green fluorescence (FITC) and red
fluorescence (PI) were detected by filtration through
FL-1 and FL-2 band pass filter, respectively. Spectral
overlap was electronically compensated using single –
color controls including cells alone, FITC alone, and
PI alone in separate tubes. Analysis of the
multivariate data was performed with Win MDI
2.8 software (Downloaded from internet). FITC- / PI-,
FITC+ / PI- or FITC+ / PI+ represented viable
(intact) cells, apoptotic cells, or necrotic cells,
respectively (Fig. 5).
Preparation of nuclear extracts—Cells treated and
irradiated in RPMI-1640 with 1% FBS (v/v) as
explained in section 2.4. Cells were incubated at
37 οC for 30 min only for NF-kB (because maximum
binding activity was observed at 30 min post
irradiation; data not shown) and used as such
immediately after irradiation for AP-1. Subsequently
washed twice with PBS, harvested and resuspended in
500 µL buffer containing 20 mM Hepes, 1.5 mM
MgCl2, 10 mM NaCl, 0.2 mM EDTA, 20% glycerol,
0.1% Triton X-100, 10 µg aprotinin, 4µg leupeptin,
0.2 M PMSF and 1 M DTT. Incubated on ice for
20 min resuspended in 100 µl of buffer containing
20 mM Hepes, 1.5 mM MgCl2, 500 mM NaCl,
0.2 mM EDTA, 20% glycerol, 0.1% Triton X-100,
1µg aprotinin, 0.4 µg leupeptin, 0.02 M PMSF and
1mM DTT. After constant agitation for 1 h at 4 οC,
nuclear debris was pelleted by centrifugation.
The supernatant was stored at -80 οC until analysis.
Electrophoretic mobility shift assay (EMSA) for
NF-kB and AP-1—EMSA was performed with 4 µg
521
nuclear protein in a total volume of 50 µL in a buffer
containing 1 M Hepes, 1 M MgCl2, 1 M NaCl, 100 %
glycerol, 1 M DTT, 0.5-µg poly [d(I-C)], 40 mM
EDTA (Ethylenediaminetetra acetic acid) and
radiolabelled NF-kB and AP-1 probes at 4 οC for 1 h.
The resultant DNA-protein complexes were resolved
from free labeled DNA by electrophoresis in non
denaturing 8% (w/v) polyacrylamide gel with
0.5 × Tris borate-EDTA electrophoresis buffer4. The
gels were subsequently dried and autoradiographed.
Statistical analysis—Values are mean of
observations made from splenocytes from 18 mice
(The experiments were carried out thrice with
6 animals in each group each time). The statistical
significance of difference between the data pairs was
evaluated by analysis of variance (ANOVA) followed
by a Mann-Whitney U-test.
Results
Effect on membrane lipid peroxidation—Lipid
peroxidation is an important effect of radiation on
biological membranes, which brings about various
changes in their structure and function42. Membrane
lipid peroxidation was found to increase with the
increase of radiation dose (0-7) Gy4 (Table 1).
To study the modulation 3 and 5 Gy of radiation and
different concentrations of Hoechst 33258 were used
(10, 20 and 30 µM). Hoechst 33258 decreases
radiation-induced (3 and 5 Gy) lipid peroxidation
(Table 2). For example, 30 µM Hoechst 33258 caused
37% and 43% decrease as compared to irradiated
control group and 20 µM Hoechst 33258 caused
31% and 35% decrease as compared to irradiated
control group at 3 Gy and 5 Gy respectively. It was
important that in unirradiated control groups, there
Table 1—Effect of different doses of γ-radiation on the levels of lipid peroxidation (LP), nitric oxide (NO.) fluidity and GSH in
splenocytes of Swiss albino mice
[Values are mean + SD of at least 3 experiments]
LP
NO.
Fluidity
GSH
(TBARS/mg protein)
(1/ polarization)
(µM)
(µmoles/mg protein)
0 Gy
0.219+0.02
0.029+0.001
4.25+0.56
0.713+0.11
(100)
(100)
(100)
(100)
3 Gy
0.306+0.03**
0.032+0.002
5.18+0.75**
0.906+0.012**
(139)
(110)
(122)
(127)
**
***
**
5 Gy
0.345+0.18
0.040+0.004
5.78+1.10
0.972+0.04**
(157)
(136)
(136)
(136)
7 Gy
0.423+0.14***
0.045+0.002***
6.10+0.28*
1.110+0.08***
(193)
(155)
(143)
(156)
Values in parentheses are % change against control. *significantly different (*P<0.001) compared with control (unirradiated with no
chemical), **P<0.05 compared with control (unirradiated with no chemical), ***P<0.005 compared with control (unirradiated with no
chemical)
Treatment
INDIAN J EXP BIOL, AUGUST 2012
522
seemed to be no adverse effect of Hoechst 33258 on
membrane lipid peroxidative damage (Table 2).
Effect on membrane fluidity—Radiation-induced
lipid peroxidation has been shown to modulate
membrane fluidity. Many membrane-dependent
processes are likely to be affected by changes in
membrane fluidity43. In view of this effect of radiation
on fluidity of membrane by using 1, 6-diphenyl-1,
3, 5-hexatriene (DPH) were checked. Immediately
after irradiation with different doses (0-7 Gy), the
splenocytes were incubated with DPH for 30 min and
fluorescence polarization was then measured as
described in earlier section (2.9). Polarization is
known to be inversely proportional to the fluidity.
Like membrane lipid peroxidation, the fluidity of
membranes also increased with increase in radiation
doses (0-7) Gy (Table 1). The presence of Hoechst
33258 during irradiation decreased the fluidity in a
concentration dependent manner (Table 2). Effect of
Hoechst 33258 on membrane fluidity is similar as its
effect on lipid peroxidation (i.e. fluidity decreased
with increase in concentration of Hoechst 33258).
Decrease in fluidity of membrane at 30 µM Hoechst
33258 caused 26% decrease at 3 Gy and
28% decrease at 5 Gy of radiation compared to
irradiated control group. Hoechst 33258 (20 µM)
caused 19% and 23% decrease at 3 Gy and 5 Gy
respectively (compared to irradiated control group).
It was important that there was not much variation in
the effect of different concentration of Hoechst 33258
in unirradiated control group of splenocytes (Table 2).
Effect on levels of nitric oxide—Nitric oxide is
endogenously formed in many biological systems and
play an important role as signaling molecule and
known to be involved in cytotoxic effect. Therefore,
in the present study, nitric oxide was determined in
terms of nitrite levels. Results showed that nitric
oxide levels were elevated progressively in
splenocytes following irradiation upto 7 Gy.
However, when splenocytes were irradiated in
Table 2—Effect of Hoechst 33258 on the levels of lipid peroxidation (LP), nitric oxide (NO.) fluidity and GSH in splenocytes of
Swiss albino mice
[Values are mean + SD of at least 3 experiments]
LP
NO.
Fluidity
GSH
(TBARS/mg protein)
(1/ polarization)
(µM)
(µmoles/mg protein)
0 Gy
0.219+0.02
0.029+0.001
4.25+0.56
0.713+0.11
(100)
(100)
(100)
(100)
0.214+0.08
0.028+0.001
4.17+0.88
0.681+0.10
+ H (10 µM)
(97)
(97)
(98)
(96)
0.209+0.03
0.027+0.002
4.06+1.67
0.586+0.01
+ H (20 µM)
(95)
(93)
(95)
(82)
0.205+0.02
0.026+0.01
3.92+1.50
0.576+0.07
+ H (30 µM)
(93)
(90)
(92)
(81)
3 Gy
0.306+0.03**
0.032+0.002
5.18+0.75**
0.906+0.012**
(139)
(110)
(122)
(127)
0.238+0.05#
0.028+0.003
4.69+0.89
0.732+0.03#
+ H (10 µM)
(108)
(96)
(110)
(103)
0.210+0.04#
0.024+0.001**#
4.19+0.98#
0.675+0.02#
+ H (20 µM)
(95)
(83)
(98)
(95)
#
**#
#
**#
0.192+0.02
0.022+0.002
3.91+0.54
0.508+0.014
+ H (30 µM)
(87)
(76)
(92)
(71)
5 Gy
0.345+0.18**
0.040+0.004***
5.78+1.10**
0.972+0.04**
(157)
(136)
(136)
(136)
0.278+0.06**
0.033+0.001#
5.07+1.02**
0.784+0.003#
+ H (10 µM)
(126)
(112)
(119)
(110)
#
r#
#
#
0.223+0.05
0.028
+
0.001
4.41+1.09
0.667+0.02
+ H (20 µM)
(101)
(96)
(103)
(94)
0.196+0.08#
0.024+0.002**#
4. 14+1.32#
0.521+0.02##**
+ H (30 µM)
(89)
(83)
(97)
(73)
Values in parentheses are the percentage change against control. *Significantly different (*P<0.001) compared with control (unirradiated
with no chemical), **P<0.05 compared with control (unirradiated with no chemical), ***P<0.005 compared with control (unirradiated with
no chemical), ##P<0.001 compared with control group (irradiated with no chemical), #P<0.05 compared with control group
(irradiated with no chemical). H = Hoechst 33258
Treatment
KHATOON & KALE: RADIOMODULATION BY HOECHST 33258
presence of different concentration of Hoechst 33258
the level of NO. decreased with increased
concentration of this compound (Table 2). For
example at 20 and 30 µM of Hoechst the percentage
decrease was found to be 25%, 31% at 3 Gy and 30%,
40% at 5 Gy of radiation respectively (compared to
irradiated control group). There was not a significant
change in the control group due to Hoechst 33258.
Effect on the antioxidant status of the cells—Since
antioxidant status is known to determine the radiation
response of cells, we have examined the effect of
different doses of radiation (0-7 Gy) on enzymes
involved in antioxidant function. Splenocytes were
irradiated with different doses (0-7 Gy). The specific
activities of DTD, SOD, GST and catalase were
enhanced at 3 and 5 Gy and then declined at 7 Gy
(Table 3). However, the levels of specific activities
at 7 Gy are still higher compared to unirradiated
controls. Hoechst 33258 was able to inhibit the
radiation-induced lipid peroxidation and membrane
fluidity. Therefore, it was interesting to see whether
Hoechst 33258 could influence the antioxidant status
of splenocytes. The splenocytes were incubated with
different concentration (10–30 µM) and irradiated at
3 or 5 Gy. The specific activities were determined
immediately after irradiation. It was significant that
Hoechst 33258 was able to enhance the specific
activities of antioxidant enzymes (Table 4).
GSH is an important aqueous–phase antioxidant
and an essential co-factor for antioxidant enzymes.
GSH plays a crucial role in scavenging the reactive
oxygen species/free radicals and in detoxification of
drugs44. On irradiation of splenocytes with 3, 5 and
7 Gy, levels of GSH were increased (Table 1). When
splenocytes were incubated in presence of Hoechst
33258 and irradiated, the levels of GSH were found to
be decreased (Table 2). Unexpectedly, Hoechst 33258
523
decreased radiation induced GSH content when it
enhanced levels of other antioxidants, while there was
not a significant change in GSH content in
unirradiated control group with Hoechst 33258.
Effect on DNA binding activity of NF-kB and
AP1—On irradiation of splenocytes, DNA binding
activity of NF-kB and AP-1 was found to be increased
with radiation dose. Effect of radiation (3 and 5 Gy)
and Hoechst 33258 was examined as shown by the
complex formation in Fig. 2. However, in presence of
Hoechst 33258 the DNA binding activity of NF-kB
was decreased (Fig. 2). Hoechst 33258 also decreased
the radiation-induced AP-1 band. The free unused
oligoprobe is indicated at the bottom of the Fig. 3.
Effect on apoptosis—Biological membranes are
considered to be critical target of radiation effect.
Recently membrane damage has been found to be a
trigger for apoptosis. Lipid peroxidation brings about
various changes in the properties of membrane
including fluidity and permeability and in turn
mediates DNA damage. Since, Hoechst 33258
inhibited radiation-induced lipid peroxidation and
membrane fluidity, an attempt has been made to
examine effect of Hoechst 33258 on radiationinduced apoptosis in splenocytes using DNA ladder
assay. Splenocytes were treated as required, irradiated
and subsequently incubated for 6 h before
electrophoresis. Presence of Hoechst 33258 during
irradiation decreased the extent of apoptosis as found
from nucleosomal ladder formation. Fig. 4 shows the
effect of Hoechst 33258 on DNA fragmentation at
3 Gy, 5 Gy of radiation and on the unirradiated
control group. Above finding were analyzed by the
densitometric measurement by using Alpha Imager
3400 programme from Alpha Innotech. It clearly
shows that Hoechst 33258 protected the cell against
undergoing apoptosis (Fig. 5).
Table 3—Effect of different doses of γ-radiation on the specific activities of antioxidant enzymes in splenocytes of Swiss albino mice
[Values are mean + SD of at least 3 experiments]
Specific activity (units/mg of protein)
Treatment
0 Gy
3 Gy
5 Gy
7 Gy
DTD
0.006+0.001
(100)
0.009+0.001*
(150)
0.010+0.003*
(166)
0.008+0.003*
(133)
GST
0.021+0.001
(100)
0.026+0.003*
(123.8)
0.045+0.003**
(214)
0.040+0.012**
(191)
SOD
1.366+0.26
(100)
1.957+0.07*
(143)
2.429+0.2**
(177)
2.026+0.13**
(148)
CAT
0.855+0.09
(100)
1.108+0.1*
(129)
1.232+0.17*
(144)
1.062+0.07*
(124)
Values in parentheses are the percentage change against control. *Significantly different (*P<0.05) compared with control
(unirradiated with no chemical), **P<0.001 compared with control (unirradiated with no chemical)
INDIAN J EXP BIOL, AUGUST 2012
524
Representive flow cytometry analysis of radiationinduced cell death is given at Fig. 6. Data from dot
plot diagrams clearly indicate that radiation-induced
apoptosis and to a lesser extent necrotic cell death is
decreased in the presence of Hoechst 33258 viz.
30 µM Hoechst 33258 caused 14% and 19%
decrease in apoptotic cells at 3 Gy and 5 Gy of
radiation respectively (compared to irradiated
control group).
Fig. 2—Effect of Hoechst 33258 on DNA binding activity of
NF-kB as assessed by EMSA using nuclear extracts of murine
splenocytes treated with Hoechst 33258 on ice for 30 min and
subsequently irradiated. Radiolabelled NF-kB oligoprobe was
incubated with the nuclear extract. Presence of Hoechst
33258 decreased the NF-kB binding activity as depicted by
decrease in complex formation. Free unused oligoprobe is seen
at the bottom of the gel. [Lane 1: 5 Gy + 30 µM H; lane
2: 5 Gy + 20 µM H; lane 3: 5 Gy + 10 µM H; lane 4: 5 Gy;
lane 5: 3 Gy + 30 µM H; lane 6: 3 Gy + 20 µM H; lane 7: 3
Gy + 10 µM H; lane 8: 3 Gy; lane 9: unirradiated control group;
H = Hoechst 33258]
Table 4—Effect of Hoechst 33258 on the specific activities of antioxidant enzymes in splenocytes of Swiss albino mice
Treatment
0 Gy
DTD
[Values are mean + SD of at least 3 experiments]
Specific activity (units/mg of protein)
GST
SOD
CAT
0.006+0.001
0.021+0.001
1.366+0.26
0.855+0.09
(100)
(100)
(100)
(100)
0.007+0.002
0.022+0.001
1.608+0.02
1.006+0.08
+ H (10 µM)
(116)
(104)
(117)
(118)
0.0075+0.003
0.023+0.002
1.710+0.74
1.028+0.5
+ H (20 µM)
(125)
(109)
(125)
(120)
0.008+0.002
0.025+0.003
1.820+0.46
1.069+0.7
+ H (30 µM)
(133)
(119)
(133)
(125)
3 Gy
0.009+0.001*
0.026+0.003*
1.957+0.07*
1.108+0.1*
(150)
(123.8)
(143)
(129)
0.010+0.002**#
0.029+0.005*
2.12+0.11**#
1.462+0.3**#
+ H (10 µM)
(166)
(138)
(168)
(171)
0.046+0.001**##
4.167+0.72**##
2.049+0.03**##
0.014+0.001***##
+ H (20 µM)
(233)
(219)
(305)
(239)
***###
**##
**###
0.017+0.001
0.051+0.004
4.414+0.1
2.241+0.13**##
+ H (30 µM)
(283)
(242.8)
(323)
(262)
0.045+0.003**
2.429+0.2**
1.232+0.17*
5 Gy
0.010+0.003*
(166)
(214)
(177)
(144)
0.018+0.001***##
0.059+0.003**#
3.046+0.6**#
1.601+0.04**#
+ H (10 µM)
(300)
(280.8)
(223)
(187)
*##
**##
**##
**#
0.019+0.002
0.063+0.003
4.237+0.5
1.687+0.01
+ H (20 µM)
(316)
(300)
(309)
(197)
0.021+0.001***##
0.078+0.001**##
5.228+0.23**###
1.838+0.03**##
+ H (30 µM)
(350)
(371)
(382)
(215)
Values in parentheses are the percentage change against control. *Significantly different (*P<0.05) compared with control (unirradiated
with no chemical), **P<0.001 compared with control (unirradiated with no chemical), ***P<0.0001 compared with control (unirradiated
with no chemical). #P<0.01 compared with control group (irradiated with no chemical), ##P<0.001 compared with control group
(irradiated with no chemical), ###P<0.0001 compared with control group (irradiated with no chemical). Abbreviations used are:H-Hoechst 33258, DTD-DT Diaphorase, GST-Glutathione S- transeferase, SOD-Superoxide dismutase, CAT-Catalase
KHATOON & KALE: RADIOMODULATION BY HOECHST 33258
525
Fig. 3—Effect of Hoechst 33258 on DNA binding activity of AP-1 as assessed by EMSA using nuclear extracts of murine splenocytes
treated with Hoechst 33258 on ice for 30 min and subsequently irradiated. Radiolabelled AP-1 oligoprobe was incubated with nuclear
extract. Presence of Hoechst 33258 decreased the AP-1 binding activity as depicted by decrease in complex formation. Free unused
oligoprobe is seen at the bottom of the gel. [Lane 1-2 (from the left): unirradiated control group; lane 3-4: 3 Gy; lane 5-6: 3 Gy + 10 µM
H; lane 7-8: 3 Gy + 20 µM H; lane 9-10: 3 Gy + 30 µM H; lane 11-12: 5 Gy; lane 13-14: 5 Gy + 10 µM H; lane 15-16: 5 Gy + 20 µM H;
lane 17-18: 5 Gy + 30 µM H; H = Hoechst 33258]
Discussion
The mechanism of protection of H-258 to DNA is
possible because of hydrogen donation, electron or
energy transfer from the ligand45. The goal of our
study was to determine the radioprotective ability of
Hoechst 33258 on membrane related signaling
pathways, particularly pathways associated with
radiation induced apoptosis. Irradiation of mice
splenocytes with different doses (0-7 Gy) resulted in a
dose-dependent increase in lipid peroxidation4
(Table 1). The radicals generated from the radiolytic
decomposition of cellular water of splenocytes might
have reacted with membranes initiating as well as
propagating the chain reaction resulting into
peroxidative damage. Hydroxyl (HO.) radical initiates
and the peroxyl (ROO.) and alkoxyl (RO.) radicals
propagate the chain reaction of peroxidation.
The removal of HO.is expected to inhibit the
propagation of radiation-induced lipid peroxidation. It
is quite clear from these findings that Hoechst 33258
probably interacted with free radicals and scavenged
them and in turn lowered the lipid peroxidation
significantly. It is also possible that Hoechst 33258
might have inactivated the free radicals of both the
types i.e. which are involved in initiating as well as in
propagation
of
lipid
peroxidation
process.
The conformational transition of the membrane
proteins are also linked with lipid peroxidation46.
In the event of the bindings of Hoechst 33258 with
receptor protein complexes of membrane may change
the conformation of both acyl chain and cis double
bonds in lipids and lead to the inhibition of
peroxidation47. Such possibility has been suggested in
case of other radiomodifiers48.
Fluidity of splenocyte membrane was increased
due to radiation (Table 1). The increase in the
membrane fluidity perhaps could be attributed to
direct effect on fatty acid chains by phospholipid
hydrolysis and therefore their increased mobility49.
Hoechst 33258 was found to inhibit the radiationinduced change in membrane fluidity of splenocytes
(Table 2). It is quite possible that scavenging of free
radicals might have resulted in an inhibition of lipid
peroxidation and in turn lowered the fluidity of
membrane. It may be mentioned that Hoechst 33258
did not affect the membrane fluidity significantly in
unirradiated control group of splenocytes. Therefore
the protective effect of Hoechst 33258 against the
526
INDIAN J EXP BIOL, AUGUST 2012
Fig. 4—Effect of Hoechst 33258 on radiation induced apoptosis
as assessed by DNA ladder formation. Presence of Hoechst
33258 caused a decrease in apoptotic DNA fragmentation.
[Lane 1: 3 Gy + 30 µM H; lane 2: 3 Gy; lane 3: 5 Gy;
lane 4: 5 Gy + 30 µM H; lane 5: 0 Gy + 30 µM H; lane 6: 0 Gy;
H = Hoechst 33258]
Fig. 5—Densitometric analysis of the apoptotic DNA ladder
formation. Preirradiation incubation with Hoechst 33258 causes
a decrease in apoptosis. [1; 0 Gy, 2; 0 Gy + 10 µM H, 3; 0 Gy +
20 µM H, 4; 0 Gy + 30 µM H, 5; 5 Gy, 6; 5 Gy + 10 µM H,
7; 5 Gy + 20 µM H, 8; 5 Gy + 30 µM H, 9; 3 Gy, 10; 3 Gy +
10 µM H, 11; 3 Gy + 20 µM H, 12; 3 Gy + 30 µM H. Alpha
Imager 3400 of Alpha Innotech, was used for densitometry
analysis. AVG is the average value of the density of each
pixel detected after background correction (AVG = IDV ÷ area);
H = Hoechst 33258].
lipid peroxidation as well as fluidity might be mainly
due to its ability to scavenge free radicals.
Nitric oxide is known to react rapidly with O2.- to
produce peroxynitrite (ONOO-) which is capable of
initiating peroxidative damage50,51. Therefore, NO. is
likely to contribute to the initiation of radiation–
induced lipid peroxidation through reactive species
like ONOO- and NO2. formed through its interaction
with O2.-. As expected, levels of NO. increased in
splenocytes on exposure to ionizing radiation
(Table 1). These findings also suggested that the
concomitant increase of lipid peroxidation and
increase in levels of NO. may also be closely
interlinked. It is also important that Hoechst 33258 was
able to lower the radiation–induced NO. formation in
splenocytes. It was quite possible that Hoechst 33258
might have also scavenged ONOO- and NO2. radicals
generated through interaction of NO. with O2.-.
Modulatory effect of Hoechst 33258 on radiationinduced lipid peroxidation, fluidity change in
membranes and generation of NO. clearly support the
idea that it has an ability to protect biological systems
against radiation effect through interference with
membrane related events. Till recently, the work has
been focused on binding of Hoechst 33258 with
DNA, almost ignoring its role as radioprotector
through modulation of membrane related events.
An important component of the signaling process
in apoptosis induced by radiation or oxidative stress/
damage suggested to be early generation of free
radicals and inturn membrane lipid peroxidation6,52,53.
Therefore irradiation, lipid peroxidation, membrane
alterations and apoptosis appears to be closely linked
and our study also supports this. Radiation induced
apoptosis in mice splenocytes was also confirmed by
flow cytometry analysis. Dot plot diagram showed the
possible occurrence of apoptosis in irradiated
splenocytes detected by Annexin V–FITC and PI
staining. It may be noted that the presence of Hoechst
33258 resulted in decreased levels of radiationinduced apoptosis. For clarity, we have measured the
density of each band (Fig. 5). These findings support
that Hoechst 33258 protects the cell from undergoing
apoptosis. Thus Hoechst 33258 is able to provide the
protection to splenocytes against radiation–induced
cell death.
Several genes have been shown to be important in
apoptosis. The modulation of expression of such
genes is mediated by transcription factors. NF-kB and
AP-1 are the the transcription factors that has been
KHATOON & KALE: RADIOMODULATION BY HOECHST 33258
527
Fig. 6—Dot Plot diagram of irradiated and unirradiated control group (5, 3, 0 Gy) and with 10, 20 and 30 µM Hoechst 33258. This
diagram represents typical apoptotic and necrotic cell population detected by Annexin V - FITC and PI staining. The percentages of
viable (intact) cells, apoptotic cells, necrotic cells are given in each quadrants of each panel. The lower left quadrant of each panel show
the viable (intact) cells which exclude PI and are negative for Annexin V–FITC binding (FITC-/PI-). The upper right quadrants contain
the non-viable, necrotic cells, positive for Annexin V –FITC binding and for PI uptake (FITC+/PI+). The lower right quadrants represent
the apoptotic cells, positive for Annexin V–FITC and negative for PI (FITC+/PI-). H-258 (Hoechst 33258).
shown to be activated by free radicals54-58. NF-kB is
stored in the cytoplasm as an inactive complex.
The signaling cascade responsible for activating
NF-kB and AP-1 is initiated at or near the plasma
membrane59, which is a site for lipid peroxidation.
AP-1 is consists of a collection of structurally related
transcription factors belonging to the jun and fos
families. Ionizing radiation stimulates the expression
of c-jun and c-fos genes. Thus, the membrane lipid
peroxidation, NF-kB, AP-1 and apoptosis might be
interlinked. Our studies also showed the involvement
of AP-1 in radiation–induced apoptosis as the result
of present study showed the increased DNA–binding
activity of NF-kB and AP-1 in irradiated splenocytes
(Figs 2 and 3). Apart from stimulating the production
of P53, the increased DNA binding activity of NF-kB
may induce transcription of specific death genes
involved in apoptosis. In the presence of Hoechst
33258 during irradiation of splenocytes resulted in
decreased DNA binding activity of NF-kB and Ap-1
(Figs 2 and 3). Scavenging of free radicals by Hoechst
33258 might have avoided the initiation of a cascade
of signaling pathways which leads to apoptotic cell
death, which is evident from the decreased DNA
binding activity of NF-kB and AP-1.
Survival of radiolytically damaged cells could
probably also depend on their ability to maintain
optimal function. The enzymes involved in the
metabolism of reactive oxygen species are likely to
play an important role in the maintenance of normal
function of cells4. Due to this, an attempt was made to
find out the radiation response of the enzymes
involved in antioxidant function. Superoxide (O.-2)
radicals formed in cells as an indirect consequence of
irradiation could be selectively scavenged by SOD.
Catalase protects the cell from H2O2, which is one of
the detrimental to biological systems and maintains
the concentration of O2 either from repeated rounds of
528
INDIAN J EXP BIOL, AUGUST 2012
chemical reduction or from the direct interaction with
toxins60. The enhanced activity of DTD might protect
the irradiated cells against free radical damage by
means of its ability to generate and maintain the
reduced antioxidant state of coenzyme Q (CoQ) in the
membranes61,62 as well as to act as two electron CoQ
reductase63. GST might catalyse antioxidant processes
of thiol compounds and protect cells from
electrophiles, free radical-induced damage and
oxidative stress29,64,65.
Enhanced activities of antioxidant enzymes are
expected to metabolize free radicals and their
products in turn protect the cells against radiation
effect. It is important that the activities of all enzymes
studied were enhanced66-69. All these enzymes are
known to function co-operatively. Our finding also
supports this aspect. Hoechst 33258 also enhanced the
specific activities of antioxidant enzymes (Table 4).
This increase is expected to contribute in
enhancement of antioxidant status of splenocytes.
Therefore, apart from scavenging the free radicals,
Hoechst 33258 seems to protect cells through
modulation of antioxidant function of cell.
GSH is important member of the non-enzymatic
antioxidant defense system. GSH has a redox
potential of around (-) 230 mv, which makes it behave
as an antioxidant and in turn efficient free radical
scavenger. It was important that the level of GSH
content of splenocytes following irradiation was
found to be increased. The level of GSH was
decreased in the presence of Hoechst 33258 and need
to be evaluated further, but there was no significant
change in unirradiated control group. Lowering of
cellular GSH content also indicates generation of
large quantity of ROS70, in this way H33258 spares
cellular GSH (both share the common property of
scavenging OH. radicals). It is important that Hoechst
33258 enhances and maintains the equilibrium of cooperative function of antioxidative enzymes. Possibly
Hoechst 33258 is itself a free radical scavenger viz.
this role is played by Hoechst-33258 (ref. 71).
Results of the present work suggested that Hoechst
33258 has an ability to protect the biological system
against radiation induced apoptosis through inhibition
of membrane related events as it reduces the lipid
peroxidation, fluidity, NO., GSH, fragmentation of
DNA, DNA binding activity of NF-kB and AP-1.
Hoechst 33258 also enhanced specific activities of
antioxidative enzymes which play an important role in
the maintenance of normal function of cells. It also
appears that radiation protection has been closely
linked with modulation of membrane related events
and elevation of antioxidant status. Earlier, the work
on Hoechst 33258 was mainly focussed on their
binding property with DNA, ignoring biochemical
pathways and membrane related events. Therefore,
the present results suggest an alternative mechanism
to explain the radioprotective potential of Hoechst
33258 to cells and tissues which are highly
radiosensitive. Examples include lymphocytes, which
are mature functional cells, have relatively high
radiosensitivity in comparison with other leukocyte
types. Radiation-induced apoptosis in lymphocytes
causes the depletion of peripheral blood lymphocytes
and leads to immunosuppression72. Therefore, it is
proposed that Hoechst 33258 treatment prior to
irradiation may have an even greater benefit in
radiotherapy since Hoechst 33258 effectively
prevented the radiation-induced apoptosis of
peripheral blood lymphocytes.
Acknowledgement
Authors gratefully acknowledge the financial
assistance of the Indian Council of Medical Research,
New Delhi, India, in the form of Senior Research
Fellowship to Zubaida Khatoon.
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